A Novel In-Frame Deletion in the Leucine Zipper Domain of C/EBP ε Leads to -Specific Deficiency

This information is current as Taizo Wada, Tadayuki Akagi, Masahiro Muraoka, Tomoko of October 1, 2021. Toma, Kenzo Kaji, Kazunaga Agematsu, H. Phillip Koeffler, Takashi Yokota and Akihiro Yachie J Immunol 2015; 195:80-86; Prepublished online 27 May 2015; doi: 10.4049/jimmunol.1402222 http://www.jimmunol.org/content/195/1/80 Downloaded from

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Material 2.DCSupplemental http://www.jimmunol.org/ References This article cites 29 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/195/1/80.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

A Novel In-Frame Deletion in the Leucine Zipper Domain of C/EBP« Leads to Neutrophil-Specific Granule Deficiency

Taizo Wada,*,1 Tadayuki Akagi,†,1 Masahiro Muraoka,* Tomoko Toma,* Kenzo Kaji,‡ Kazunaga Agematsu,x H. Phillip Koeffler,{,‖ Takashi Yokota,† and Akihiro Yachie*

Neutrophil-specific granule deficiency (SGD) is a rare autosomal recessive primary immunodeficiency characterized by neutrophil dysfunc- tion, bilobed neutrophil nuclei and lack of neutrophil-specific granules. Defects in a myeloid-specific transcription factor, CCAAT/enhancer binding protein-« (C/EBP«), have been identified in two cases in which homozygous frameshift mutations led to loss of the leucine zipper domain. In this study, we report a 55-y-old woman affected with SGD caused by a novel homozygous 2-aa deletion (DRS) in the leucine zipper domain of the C/EBP« . The patient showed characteristic neutrophil abnormalities and recurrent skin infections; however, D there was no history of deep organ infections. Biochemical analysis revealed that, in contrast to the two frameshift mutations, the RS Downloaded from mutant maintained normal cellular localization, DNA-binding activity, and dimerization, and all three mutants exhibited marked reduction in transcriptional activity. The DRS mutant was defective in its association with Gata1 and PU.1, as well as aberrant cooperative transcrip- tional activation of . Thus, the DRS likely impairs protein-protein interaction with other transcription factors, resulting in a loss of transcriptional activation. These results further support the importance of the leucine zipper domain of C/EBP« for its essential function, and indicate that multiple molecular mechanisms lead to SGD. The Journal of Immunology, 2015, 195: 80–86. http://www.jimmunol.org/ eutrophil-specific granule deficiency (SGD) is a rare au- in elevated levels of C/EBPε (4). In this patient, the growth fac- tosomal recessive primary immunodeficiency characterized tor independence 1 (Gfi-1) protein that represses transcription of N by either profound reduction or absence of neutrophil- C/EBPε was decreased, although the patient had no mutation of specific granules and bilobed neutrophil nuclei (pseudo–Pelgar- the Gfi-1 gene. Taken together, these previous findings suggest Hue¨t anomaly) (1). It was previously called “ deficiency.” that SGD is a genetically heterogeneous disease. Because of the Patients with SGD exhibit increased susceptibility to bacterial in- extreme rarity of SGD, the full spectrum of clinical symptoms and fections, especially affecting the skin, ears, lungs, and lymph nodes. cellular abnormalities of the disease is unclear. The gene responsible for SGD is the CCAAT/enhancer binding C/EBPε is a member of the C/EBP family of widely expressed protein-ε (C/EBPε) gene. To date, two patients have been reported transcription factors that regulate proliferation, differentiation, and by guest on October 1, 2021 who carry C/EBPε frameshift mutations that result in abrogated apoptosis in a variety of cell types (5, 6). The C/EBP family consists protein expression (2, 3). In addition, another patient with SGD had of six members (C/EBPa, b, g, d, ε,andz), and cellular expression a heterozygous missense mutation of the C/EBPε gene, but this of each C/EBP is tightly regulated. They bind to DNA through the mutation was unlikely to have caused disease because it resulted highly conserved basic leucine zipper (bZIP) domain. C/EBPε is restricted to and is essential for their terminal differ- entiation (6, 7). C/EBPε transcription primarily occurs at the mye- *Department of Pediatrics, Institute of Medical, Pharmaceutical and Health Sci- locyte–metamyelocyte stage of differentiation and decreases in ences, School of Medicine, Kanazawa University, Kanazawa 920-8641, Japan; polymorphonuclear . The human C/EBPε gene produces †Department of Stem Cell Biology, Institute of Medical, Pharmaceutical and Health Sciences, School of Medicine, Kanazawa University, Kanazawa 920-8640, Japan; four isoforms of 32, 30, 27, and 14 kDa that are functionally dif- ‡Department of Dermatology, Komatsu Municipal Hospital, Komatsu 923-0961, ferent; only the 32-kDa C/EBPε has full transactivating potential (8– Japan; xDepartment of Infection and Host Defense, Shinshu University Graduate School { 10). C/EBPε is indispensable for expression of encoding of Medicine, Matsumoto 390-8621, Japan; Division of Hematology and Oncology, Cedars-Sinai Medical Center, University of California Los Angeles School of Med- proteins that reside in specific granules of neutrophils, such as lac- icine, Los Angeles, CA 90048; and ‖Cancer Science Institute of Singapore, National toferrin and . Many features of SGD are manifested by University of Singapore, Singapore 117599, Singapore C/EBPε-deficient mice in which neutrophils are morphologically and 1 T.W. and T.A. contributed equally to this work. functionally abnormal and eosinophil numbers are decreased (8). Received for publication September 8, 2014. Accepted for publication May 4, 2015. In this study, we describe another case of SGD with a novel 2-aa This work was supported by a Grant-in-Aid for Scientific Research from the Ministry deletion in the bZIP domain of C/EBPε, and we report the mech- of Education, Culture, Sports, Science and Technology of Japan and a grant from the anism that leads to SGD. We also completed clinical, cellular, and Ministry of Health, Labor, and Welfare of Japan, Tokyo. molecular comparisons for this patient and the previously reported Address correspondence to Dr Taizo Wada, Department of Pediatrics, School of Medicine, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa Uni- two cases of SGD (2, 3), and we discuss the functional significance versity, 13-1 Takaramachi, Kanazawa 920-8641, Japan. E-mail address: taizo@staff. of the mutation. kanazawa-u.ac.jp The online version of this article contains supplemental material. Abbreviations used in this article: bZIP, basic leucine zipper; C/EBPε, CCAAT/enhancer Materials and Methods binding protein-ε; Gata1, GATA binding protein 1; MaBP, major basic protein; MBP, Patients maltose-binding protein; NGAL, neutrophil gelatinase-associated lipocalin; Prg2, pro- teoglycan 2; SGD, neutrophil-specific granule deficiency; WT, wild-type. We studied two Japanese patients with SGD. Patient P1 is a 55-y-old woman who has suffered since late infancy from recurrent skin infections that often Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 required more than 2 mo to heal. After hospitalization because of severe www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402222 The Journal of Immunology 81 otitis media at 54 y of age, she was referred to our hospital for suspec- Biotin-labeled DNA pull-down assay, MBP pull-down assay, ted immunodeficiency. A history of parental consanguinity appeared likely. and Western blot analysis However, her father had already died of a heart attack, and her mother refused genetic analysis. Her elder brother had similar skin symptoms and died of Biotin-labeled DNA pull-down assay was performed as described previ- enterocolitis at 10 y of age. Another brother also died early after birth from ously (13, 17). Briefly, biotin-labeled oligonucleotides containing the human unspecified causes. Two children of patient P1 and her granddaughter were lactoferrin gene C/EBPε binding site (59-GGGTGTCTATTGGGCAACA- healthy, and they did not want genetic testing. Clinical and genetic data of GGGCGGG-39) were incubated with cell extracts from HEK293 cells patient P2 have already been published (3, 11). Patient P2 is now 40 y old. transfected with either pCAGIP-Myc-C/EBPε or its mutant counterparts Approval for the study was obtained from the Human Research Committee (DRS, del5bp, and insA) in the presence of streptavidin-agarose (Novagen, of Kanazawa University Graduate School of Medical Science, and informed Darmstadt, Germany). Twenty-five-fold nonlabeled oligonucleotides (either consent was provided according to the Declaration of Helsinki. WT or mutant nonbinding control) were added for the competition assays. The beads were washed three times with a washing buffer, and the bound Flow cytometry and immunohistochemistry proteins were eluted by boiling in 2X SDS sample buffer. Samples were then examined by Western blot analysis as described below. MBP pull-down For flow cytometric analysis, the following mAbs were used: FITC- assay was performed as described previously (13, 17). HEK293 cells were conjugated anti-CD16, anti-CD32 or CD66b and PE-conjugated anti- cotransfected with pCAGIP-Myc-C/EBPε (WT or DRS) and pCMV5-Flag- CD11b, anti-CD14, anti-CD15 anti-CD16 or anti-CD64 (BD, San Diego, MBP-C/EBPε (WT or DRS). HEK293 cells were also cotransfected with CA); and FITC-conjugated anti-CD16b and PE-conjugated CD11c (Beckman pCAGIP-Myc-Gata1 and pCMV5-Flag-MBP-C/EBPε or its mutant coun- Coulter, Fullerton, CA). Analysis was performed on a FACSCalibur using terparts, or with pCAGIP-Myc-PU.1 and pCMV5-Flag-MBP-C/EBPε or its CellQuest software (BD Biosciences, Tokyo, Japan) (12). Cytospin prepa- mutant counterparts. MBP-fused proteins were pulled down by amylose rations were made using whole leukocytes. For lactoferrin staining, cytospin resin, and the precipitates were analyzed by Western blot analysis. For samples were stained with anti-lactoferrin mAb (Beckman Coulter), fol- Western blot analysis, samples were subjected to SDS-PAGE and transferred lowed by universal immuno-alkaline-phosphatase polymer (Simple Stain to a nitrocellulose membrane. The membrane was incubated with either

AP; Nichirei Biosciences, Tokyo, Japan). The activity Downloaded from anti-Myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA) or anti-Flag was visualized using Fast Red TR salt and Naphtol AS-MX phosphate (F3165; Sigma-Aldrich) Abs followed by HRP-conjugated anti-mouse Ab (Sigma-Aldrich, St. Louis, MO). For analysis of internal alkaline phospha- (Millipore, Billerica, MA). The blot was visualized using ECL reagents tase and peroxidase activity, alkaline phosphatase substrate solution (BCIP/ (PerkinElmer, Waltham, MA) with an LAS-1000 image analyzer (Fuji Film, NBT Substrate System; DAKO, Glostrup, Denmark) and a peroxidase stain- Tokyo, Japan). ing kit (Muto Pure Chemicals, Tokyo, Japan) were used, respectively. Mutation analysis Results http://www.jimmunol.org/ Peripheral blood or buccal mucosa samples, or both, were obtained from Bilobed nucleus and lack of granules in neutrophils patients, and DNAwas extracted from the samples using a standard method. Patient P1 exhibited an ulcerative skin abscess and numerous skin Direct sequencing of the C/EBPε gene was performed as described pre- viously (2). scars (Fig. 1A, 1B). Her peripheral blood smear showed unique bilobed nuclei and a lack of cytoplasmic granules in her neutrophils Cell culture and RT-PCR (Fig. 1C). Absence of , increased with normal Human embryonic kidney (HEK) 293 and mouse NIH3T3 cells were cultured cytoplasmic granularity, and monocytosis were also noted on in DMEM containing either 10% FBS or 10% calf serum, respectively. Total smears of the peripheral blood (data not shown). The immunohis- RNAwas extracted from transfected NIH3T3 cells with Sepasol-RNA I Super tochemical analysis of her neutrophils clearly demonstrated the G (Nacalai Tesque, Kyoto, Japan) and converted to cDNAs by ReverTraAce (Toyobo, Tokyo, Japan). Expression of human C/EBPε and PU.1, and murine presence of peroxidase, a primary granule protein, and the absence by guest on October 1, 2021 B9/neutrophil granule protein, neutrophil gelatinase-associated lipocalin (NGAL)/ lipocalin 2, lactoferrin, 2 (Prg2)/eosinophil granule eosinophil major basic protein (MaBP), GATA binding protein 1 (Gata1), and GAPDH was examined by RT-PCR. Primer sequences are re- ported in Supplemental Table I. The number of amplification cycles was 20 for GAPDH; 25 for C/EBPε, PU.1, MaBP, and Gata1; 30 for B9 and NGAL; and 35 for lactoferrin. The PCR cycle consisted of 94˚C for 15 s, 60˚C for 30 s, and 72˚C for 30 s. For lactoferrin, the annealing temperature was 64˚C. All PCR reactions were completed with Taq polymerase (Ampliqon, Odense, Denmark) except for C/EBPε (PrimeSTAR Max DNA Polymerase; Takara Bio, Otsu, Japan). Construction of plasmid, luciferase assay, and cellular localization Construction of mammalian expression vectors, including pCMV5-Flag maltose-binding protein (MBP), pCAGIP-enhanced GFP, and pCAGIP- Myc, was described previously (13, 14). Coding region of wild-type (WT) human C/EBPε and its mutants, DRS, del5bp, and insA, were amplified by PCR using specific primers (Supplemental Table I) and cloned into ex- pression vectors. Murine Gata1 and human PU.1 expression vectors were described previously (15), and their coding regions were cloned into the pCAGIP-Myc vector. The luciferase reporter plasmid containing the G-CSF receptor promoter (pGCSFR-Luc) has been described previously (16). The reporter plasmid, C/EBPε and C/EBPε mutant expression vectors (DRS, del5bp, and insA), were transfected into HEK293 cells by Lipofectamine 2000 (Life Technologies, Grand Island, NY). To determine luciferase ac- tivity, cell extracts were prepared 48 h after transfection, and luciferase ac- tivity was measured with a luciferase assay kit (Promega, Madison, WI) FIGURE 1. Patient characteristics. (A and B) Skin abscess with ulcer- using an AB-2200 luminometer (ATTO, Tokyo, Japan). Protein concentra- ation and scar. (C) Peripheral blood smears. Neutrophils from patients P1 tion of each sample was measured by Protein Assay CBB solution (Nacalai € Tesque). Amounts of cell extracts used to obtain relative luciferase activities and P2 have bilobed nuclei and lack cytoplasmic granules (May–Grune- 3 D were normalized to 1 mg total protein per sample. To observe cellular lo- wald–Giemsa staining). Original magnification 400. ( ) Immunohis- calization of C/EBPε and its mutants, GFP-tagged fusion protein expression tochemial analysis. Cytospin preparations of leukocytes stained with anti- vectors were introduced into NIH3T3 cells. Nuclei were stained by Hoechst lactoferrin mAb. Cellular alkaline phosphatase and peroxidase activity 33258 (Sigma). Signals were observed 48 h after transfection. were also analyzed. Original magnification 3400. 82 NOVEL C/EBPε DELETION MUTATION IN SGD of lactoferrin and alkaline phosphatase, both of which are normally Fig. 4A, WT C/EBPε presented robust reporter activity, whereas the expressed in specific granules of normal neutrophils (Fig. 1D). DRS mutant exhibited a significant decrease in activity, similar to the These characteristic features were similar to those of patient P2, the previously reported mutants, insA and del5bp (c.249_253delTGACC) second case of genetically defined SGD, who carries a homozygous (2, 3), both of which were frameshift mutations with truncated pro- C/EBPε mutation, c.508_509insA (insA) (3). teins. HEK 293 cells transfected with the DRS mutant expressed Flow cytometric analysis of peripheral blood showed lower side levels of C/EBPε comparable to wild-type, as assessed with Western scatter of P1 neutrophils compared with normal controls (Fig. 2) blot analysis, indicating DRS does not cause instability in the mutated (18). More importantly, the majority of the patient’s neutrophils that C/EBPε protein (Fig. 4B). Increasing amounts of DRS had no neg- were defined on the basis of forward and side scatter expressed ative effect on reporter activity of WT C/EBPε, indicating a lack of a a monocyte marker CD14, indicating aberrant development toward dominant-negative effect of DRS on transcriptional activity (Fig. 4C). the monocyte pathway. These cells did not express neutrophil We also analyzed the ability of the C/EBPε mutant proteins to acti- markers such as CD15, CD16b (FcgRIIIB), and CD66b. Although vate gene expression of secondary granule genes. As shown in CD64 (FcgRI) was not detected on her neutrophils and monocytes, Fig. 4D, WT C/EBPε was able to induce expression of endogenous CD11b, CD11c, and CD32 (FcgRII) were detectable equally on B9, NGAL, and lactoferrin genes in transiently transfected NIH3T3 both cells (data not shown). Again, all these characteristic features cells. In contrast, none of these genes was amplified from cells were similar to those of patient P2. Neutrophils from patient P2 transfected with either the DRS mutant or the insA and del5bp showed more CD16 expression compared with P1. mutants, consistent with loss of their capability to activate secondary granule genes. 2-aa deletion in bZIP and reduced transcriptional activity

Direct sequence analysis revealed that patient P1 had a homozygous Unaltered cellular localization, DNA-binding, and Downloaded from 6-bp deletion in exon 2 of the C/EBPε gene (c.739_744delCGCAGC). dimerization This novel mutation leads to a 2-aa deletion, p.Arg247_Ser248del To understand the mechanism by which the DRS mutation decreases (DRS), which is located in the bZIP domain (Fig. 3). The mutation transcriptional activity, we first investigated the cellular localization was present in DNA isolated from both the peripheral blood and of WT and mutant C/EBPε proteins. GFP fluorescence was dif- buccal mucosa, indicating a germline mutation, not a somatic one. fusely detected within the cytoplasm of NIH3T3 cells transfected

Analysis of 100 alleles of ethnically matched healthy controls dem- with the control GFP vector, whereas WT and DRS mutant C/EBPε http://www.jimmunol.org/ onstrated the absence of the mutation in the general population. tagged with GFP was found in the nucleus (Fig. 5). Consistent with To evaluate the transcriptional activation by the DRS mutant, HEK293 cells were cotransfected with a G-CSF receptor promoter reporter construct and C/EBPε expression vectors. As shown in by guest on October 1, 2021

FIGURE 3. Mutation analysis of C/EBPε gene. (A)C/EBPε gene exon 2 was amplified from DNA extracted from normal control, as well as pe- ripheral blood and buccal mucosa of patient P1. Direct sequencing was performed using an automated sequencer. A thick bar highlights the position of the 6-bp deletion (DRS). (B) Predicted structures of mutated C/EBPε molecules. The previously reported frameshift mutations, del5bp and insA, FIGURE 2. Characterization of granulocytes. Granulocytes were gated for produced frameshifts that result in incorrect amino acid sequence subsequent fluorescence analysis. The percentage of cells gated in each region is shown. to the mutations (shaded area) and premature termination. The Journal of Immunology 83

FIGURE 4. Transcriptional activity and induction of endogenous expression of granule genes. (A) Lucifer- ase reporter plasmid containing the G-CSF receptor promoter (pGCSFR-Luc) was transfected into HEK293 cells with either control empty (ev), WT, DRS, del5bp, or insA C/EBPε expression vector. Luciferase activity was measured 48 h after transfection. Bars represent the means and SDs of triplicate assay. (B) Western blot analysis of C/EBPε was performed using lysates of HEK293 cells transfected with ev, WT, DRS, del5bp or insA C/EBPε vector. (C) WT C/EBPε vector was mixed with increasing amounts of DRS C/EBPε vector, and luciferase reporter assay was performed. (D) Ex- pression vectors of ev, WT, DRS, del5bp, or insA C/EBPε were transfected into NIH3T3 cells, and ex- pression of endogenous B9, NGAL, and lactoferrin was

examined by RT-PCR. GAPDH was used as a loading Downloaded from control. The numbers of PCR cycles were 20 for GAPDH; 30 for C/EBPε, B9, and NGAL; and 35 for lactoferrin. http://www.jimmunol.org/ the previous report (3), the insA mutant was localized in the cyto- C/EBPε in lysates of HEK293 cells transfected with the WT Flag- plasm and the nucleus; and a similar abnormal localization was MBP-C/EBPε vector, and no binding was observed in lysates of detected in the del5bp. cells transfected with the del5bp and the insA mutants (Fig. 7B, We next assessed the ability of the WT and mutant C/EBPε to 7C). Some binding of the DRS mutant to Gata1 or PU.1 was in- bind to C/EBPε binding site at the 59UTR of human lactoferrin dicated by the results, but the amounts of precipitated Gata1 and gene (19). The oligonucleotides containing the C/EBPε binding PU.1 were extremely low in lysates from HEK293 cells trans- ε

site bound in vitro to C/EBP from lysates of HEK293 cells fected with the DRS mutant. by guest on October 1, 2021 transfected with WT Myc-C/EBPε vector (Fig. 6A, top left blot). Importantly, the DRS mutant showed DNA-binding ability com- Discussion parable to the WT C/EBPε (Fig. 6A, top right blot). Nonlabeled C/EBPε is essential for terminal differentiation of granulocytes. oligonucleotides of the same sequence, but not those with a mu- Frameshift mutations of the C/EBPε gene have been identified in tated sequence, were able to compete with the biotinylated oli- two patients with SGD. In this study, we report on a 55-y-old gonucleotides for sequence-specific binding of both the WT and woman (P1) affected with SGD caused by a novel 2-aa deletion DRS mutant C/EBPε. In contrast, no product was precipitated mutation of the C/EBPε gene. This case represents a third case of from lysates of cells transfected with the del5bp and the insA genetically defined SGD. The availability of blood samples from mutants, indicating that no oligonucleotide binding took place the previous case (P2) offered us the unique opportunity to eval- (Fig. 6A, bottom panels). uate and compare phenotype of peripheral neutrophils in these To assess further the functionality of the DRS mutant, we ex- patients. In addition to the morphologic abnormalities typical for amined dimer formation in lysates of HEK293 cells cotransfected SGD, we found characteristic surface phenotype in their neutrophils, with Myc-C/EBPε and Flag-MBP-C/EBPε vectors. The DRS mutant was able to homodimerize or heterodimerize with WT C/ EBPε at levels comparable to WT C/EBPε (Fig. 6B and data not shown). These results were consistent with the ability of the DRS mutant to bind to DNA. Aberrant association with Gata1 and PU.1 To determine whether the DRS mutant properly interacts with other proteins, cooperative transcriptional activation of MaBP was analyzed in NIH3T3 cells. Consistent with the previous report (15), MaBP gene expression was observed in cells transfected with Gata1 and PU.1 in addition to WT C/EBPε, and no products were obtained from those without WT C/EBPε (Fig. 7A). Inter- estingly, the DRS mutant, as well as the del5bp and the insA FIGURE 5. Cellular localization. GFP-tagged to either control empty mutants, failed to induce MaBP gene expression. (ev), WT (WT), DRS, del5bp or insA C/EBPε expression vectors were To assess further the ability of the C/EBPε mutants to bind to transfected into NIH3T3 cells, and cells were analyzed by microscopy and Gata1 as well as PU.1, the MBP pull-down assay was performed. fluorescent microscopy 2 d after transfection. Nuclei were stained with As expected, both Gata1 and PU.1 were able to bind in vitro to Hoechst stain. Scale bar, 20 mm. 84 NOVEL C/EBPε DELETION MUTATION IN SGD Downloaded from

FIGURE 6. DNA binding activity and dimerization. (A) DNA binding activity of WT, DRS, del5bp, and insA C/EBPε. Biotin-labeled oligonucleotide containing the C/EBPε-binding site of the lactoferrin gene was incubated with Myc-C/EBPε (WT, DRS, del5bp, or insA)-transfected HEK293 cell extracts either with or without 25-fold nonlabeled WT or mutated nonbinding (m) oligonucleotide. Biotin-labeled oligonucleotides were pulled down by strepta- vidin-agarose. The precipitates and cell lysates were analyzed by Western blot analysis with anti-Myc Ab. (B) Dimer formation of DRS. HEK293 cells were http://www.jimmunol.org/ transfected with Myc-C/EBPε-DRS together with either empty control vector (MBP-ev), Flag-MBP-C/EBPε-WT or -DRS. MBP-fused proteins were pulled down by amylose resin, and the precipitates were analyzed by Western blot analysis with anti-Myc Ab. Expression of each protein was confirmed with anti- Myc and anti-Flag Abs, respectively. including the presence of monocyte markers such as CD14 and the whereas CD16b is a GPI-anchored receptor that is thought to be absence of neutrophil markers such as CD15, CD16b, and CD66b. expressed exclusively by neutrophils. Because pan-CD16 but not It is therefore difficult to distinguish neutrophils from monocytes CD16b was detected in a subset of the patient’s neutrophils, they by surface markers in the patients. CD16 includes two isoforms, likely expressed CD16a. Neutrophils from patient P2 showed higher by guest on October 1, 2021 CD16a (FcgRIIIA) and CD16b. CD16a is a transmembrane receptor levels of CD16 expression than those of patient P1 did, indicating expressed by monocytes, natural killer cells and natural killer T cells, a larger subpopulation of CD16a+ neutrophils. Human monocytes

FIGURE 7. Cooperative transcriptional activa- tion of eosinophil major basic protein (MaBP). (A) Induction of Prg2/MaBP expression in NIH3T3 cell. Expression vectors Gata1 and PU.1 were transfected into NIH3T3 cells either with or without C/EBPε expression vectors (WT, DRS, del5bp, or insA). Expression of endogenous Prg2/MaBP mRNA was examined by RT-PCR. GAPDH was used as a load- ing control. The numbers of PCR cycles were 20 for GAPDH, and 25 for C/EBPε, Gata1, PU.1 and MaBP. (B and C) Protein interactions between C/EBPε WT or mutants with either Gata1 or PU.1. HEK293 cells were transfected with either Myc- Gata1 or Myc-PU.1 together with an empty control vector (MBP-ev), Flag-MBP-C/EBPε-WT, -DRS, -del5bp, or -insA. MBP-fused proteins were pulled down by amylose resin, and the precipitates were analyzed by Western blot analysis with anti-Myc Ab. Expression of each protein was confirmed with anti- Myc and anti-Flag Abs, respectively. The Journal of Immunology 85 are divided into two major subsets: CD14++CD16a2 and CD14+ infection, whereas the other patients exhibited more severe pre- CD16a+ cells. Various inflammatory conditions including infections sentation of the disease. The first reported patient with the del5bp lead to an increased subpopulation of CD16a+ monocytes (20, 21). mutation died of complications of pneumonia, and patient P2 suf- CD16 expression of neutrophils of patient P1 was associated with fered from recurrent pneumonia, as well as lung abscess (2, 3). On infections (data not shown); therefore, CD16a expression on SGD the other hand, recurrent bacterial skin abscess that persisted a few neutrophils may depend on inflammatory immune stimuli. months was observed in all patients with SGD, including patient P1 The reason why the patient’s neutrophils expressed monocyte (27). Skin abscess smears from patient P1 showed that most infil- markers, including CD14 and CD16a, is presently unclear. Studies trating cells were monocytes and , some of which of granulocytes from healthy volunteers who were given G-CSF phagocytosed bacteria (data not shown). Monocytes from C/EBPε- and from human embryonic stem cells treated with multiple deficient mice exhibited impaired maturation and altered cytokine growth factors have demonstrated aberrant expression of CD14 on expression, such as increased levels of TNF-a and LTb, in response mature granulocytes (22, 23). Although we did not measure any to inflammation (28, 29). In addition, monocyte counts in C/EBPε- soluble factors related to granulocytic differentiation in our deficient mice were higher than those of WT mice (30). Thus, im- patients, defective myeloid differentiation in SGD could lead to pairedinflammatoryresponseandkillingofbacteriabySGD dysregulated secretion of growth factors resulting in aberrant patients’ monocytes can hinder the healing process, resulting in surface expression of neutrophil proteins. In vitro modeling of unique skin abscesses. Understanding which factors evoke an ab- neutrophil development in SGD using induced pluripotent stem normal microenvironment at infectious sites will be necessary to cells will be required to address these issues. develop more effective therapeutic approaches for patients with SGD. We carried out a comprehensive in vitro study to evaluate In summary, our studies identified a novel in-frame deletional transcriptional activity, cellular localization, DNA-binding activity, mutation in the bZIP domain of C/EBPε and demonstrated its Downloaded from dimerization and protein-interaction of the DRS mutant as well as molecular pathogenesis leading to SGD. Comparative analysis of the two frameshift mutants. All three mutants exhibited marked the C/EBPε mutations, including the previous frameshift muta- reduction in transcriptional activity. The DRS mutation is located tions, also clarifies the functional significance of these mutants. in the bZIP domain, which is highly conserved among the C/EBP Characterization of C/EBPε genetic defects and functional ab- family members and has an important role in DNA binding and normalities will help to define the role of C/EBPε in human dimerization (5). However, the DRS mutant maintained normal myelopoiesis and innate immunity. http://www.jimmunol.org/ cellular localization, DNA-binding activity, and dimerization, in contrast to the frameshift mutations del5bp and insA, which de- Acknowledgments stroy the bZIP domain and thus are predicted to interfere with We thank Dr. Kuniaki Naganuma for patient care and Harumi Matsukawa dimerization and binding to DNA. No dominant-negative effect of and Shizu Kouraba for technical assistance. the DRS mutant may suggest that the single normal C/EBPε allele is sufficient to maintain transcriptional activity, which is consistent Disclosures with the fact that the mother and two children of patient P1, who The authors have no financial conflicts of interest. are assumed to be heterozygous for the DRS mutation, remain in by guest on October 1, 2021 good health. The association of C/EBPε with other transcription factors has been demonstrated to be important for the regulation of References secondary granule gene expression in both neutrophils and eosi- 1. Gombart, A. F., and H. P. Koeffler. 2002. Neutrophil specific granule deficiency nophils (15). In fact, the DRS mutant was found to be defective in and mutations in the gene encoding transcription factor C/EBP(ε). Curr. Opin. association with Gata1 and PU.1, as well as aberrant cooperative Hematol. 9: 36–42. 2. Lekstrom-Himes, J. A., S. E. Dorman, P. Kopar, S. M. Holland, and J. I. Gallin. transcriptional activation of eosinophil MaBP. Gata1 is primarily 1999. Neutrophil-specific granule deficiency results from a novel mutation with associated with erythroid and megakaryocyte differentiation, loss of function of the transcription factor CCAAT/enhancer binding protein ε. whereas PU.1 is more important for neutrophil differentiation. J. Exp. Med. 189: 1847–1852. 3. Gombart, A. F., M. Shiohara, S. H. Kwok, K. Agematsu, A. Komiyama, and These results are in line with the fact that eosinophils were not H. P. Koeffler. 2001. Neutrophil-specific granule deficiency: homozygous re- detectable in patients P1 and P2. Taken together, our findings cessive inheritance of a frameshift mutation in the gene encoding transcription ε suggest that the DRS mutation impairs protein-protein interaction factor CCAAT/enhancer binding protein— . Blood 97: 2561–2567. 4. Khanna-Gupta, A., H. Sun, T. Zibello, H. M. Lee, R. Dahl, L. A. Boxer, and with Gata1 and with PU.1, resulting in loss of cooperative tran- N. Berliner. 2007. Growth factor independence-1 (Gfi-1) plays a role in medi- scriptional activation. ating specific granule deficiency (SGD) in a patient lacking a gene-inactivating A similar mutation has been described in a patient with acute mutation in the C/EBPepsilon gene. Blood 109: 4181–4190. 5. Tsukada, J., Y. Yoshida, Y. Kominato, and P. E. Auron. 2011. The CCAAT/ myeloid leukemia, in which an in-frame 3-bp deletion within the enhancer (C/EBP) family of basic-leucine zipper (bZIP) transcription factors leucine zipper domain of C/EBPa abrogated the transcriptional is a multifaceted highly-regulated system for gene regulation. Cytokine 54: 6–19. 6. Chumakov, A. M., I. Grillier, E. Chumakova, D. Chih, J. Slater, and activation function of C/EBPa on the G-CSF receptor promoter H. P. Koeffler. 1997. Cloning of the novel human myeloid-cell-specific C/EBP-ε (24). Like the DRS mutant, this mutant lacked a dominant-negative transcription factor. Mol. Cell. Biol. 17: 1375–1386. effect, although its protein-protein interaction with other transcrip- 7. Lekstrom-Himes, J. A. 2001. The role of C/EBP(ε) in the terminal stages of differentiation. Stem Cells 19: 125–133. tion factors remained unexamined. Further investigation will be 8. Yamanaka, R., C. Barlow, J. Lekstrom-Himes, L. H. Castilla, P. P. Liu, M. Eckhaus, necessary to assess whether the DRS mutant also exhibits defective T. Decker, A. Wynshaw-Boris, and K. G. Xanthopoulos. 1997. Impaired gran- interaction with other transcription factors such as c-Myb, PML, ulopoiesis, myelodysplasia, and early lethality in CCAAT/enhancer binding protein ε-deficient mice. Proc. Natl. Acad. Sci. USA 94: 13187–13192. p300, E2F1, and Rb (9, 16, 25, 26). Because certain isoforms of 9. Verbeek, W., A. F. Gombart, A. M. Chumakov, C. Muller,€ A. D. Friedman, and C/EBPε have been reported to inhibit the synergistic activities of H. P. Koeffler. 1999. C/EBPepsilon directly interacts with the DNA binding GATA1 and PU.1 (25), we also need to evaluate the isoforms of the domain of c-myb and cooperatively activates transcription of myeloid promoters. ε ε Blood 93: 3327–3337. C/EBP mutants other than the full-length, 32-kDa C/EBP . 10. Tang, J. G., and H. P. Koeffler. 2001. Structural and functional studies of These characteristics of the DRS mutant, wherein modest as- CCAAT/enhancer-binding protein epsilon. J. Biol. Chem. 276: 17739–17746. sociation with Gata1 and PU.1 is retained and nuclear localization 11. Shiohara, M., A. F. Gombart, Y. Sekiguchi, E. Hidaka, S. Ito, T. Yamazaki, H. P. Koeffler, and A. Komiyama. 2004. Phenotypic and functional alterations of remains intact, may be associated with less severe clinical symp- peripheral blood monocytes in neutrophil-specific granule deficiency. J. Leukoc. toms of patient P1. 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